Electrogenic exchange of Na+ and K+ by the Na,K-ATPase is responsible for establishing ion concentration gradients across cell membranes that underlie electrical excitability, solute transport and volume regulation in almost all mammalian cells. In spite of this enzyme's central role in cell physiology, many questions about its mechanism of ion transport remain unanswered, including the mechanism of membrane potential (VM)- dependent extracellular ion binding and occlusion. Even recent crystallographic data provided by a related ion transport ATPase, SERCA1a, does not answer this question because its lumenal ion release pathway (equivalent to an extracellular pathway in the Na,K-ATPase) is not evident in high resolution structural models. Thus, the goal of this project is to determine the molecular mechanism and structural features of the Na,K-ATPase that underlie VM-dependent extracellular ion binding and occlusion by the Na,K-ATPase. To achieve this goal, a combination of electrophysiological, biochemical and molecular biological techniques will be used to investigate the hypothesis that transmembrane helices and extracellular loops of the enzyme's alpha-subunit create an access channel that permits VM-dependent extracellular K+ (K+o) binding and occlusion. Proposed experiments will take advantage of our recent findings and preliminary data that show the organic quaternary amine benzyltriethylammonium ion (BTEA) inhibits Na,K-pump current by binding at or near VM-dependent K+o binding sites in the Na,K-ATPase, even though the amine does not itself become occluded by the enzyme. We propose to use BTEA, and other structurally-related quaternary amines that we synthesize, to determine the mechanism by which quaternary amines gain access to K+o binding sites in the enzyme. Synthesized quaternary amines containing a methanethiosulfonate moiety will then be used to identify specific amino acid residues that are accessible during inhibition of enzymes carrying Cys mutations in transmembrane helices and extracellular loops of the alpha-subunit. The resulting accessibility data will be combined with structural data for SERCA1a (and Na,K-ATPase if it's available) to develop a map of the enzyme structures that serve extracellular ion binding and occlusion by the Na,K-ATPase.